Ail Proteins of Yersinia pestis and Y. pseudotuberculosis Have Different Cell Binding and Invasion Activities

The Yersinia pestis adhesin Ail mediates host cell binding and facilitates delivery of cytotoxic Yop proteins. Ail from Y. pestis and Y. pseudotuberculosis is identical except for one or two amino acids at positions 43 and 126 depending on the Y. pseudotuberculosis strain. Ail from Y. pseudotuberculosis strain YPIII has been reported to lack host cell binding ability, thus we sought to determine which amino acid difference(s) are responsible for the difference in cell adhesion. Y. pseudotuberculosis YPIII Ail expressed in Escherichia coli bound host cells, albeit at ∼50% the capacity of Y. pestis Ail. Y. pestis Ail single mutants, Ail-E43D and Ail-F126V, both have decreased adhesion and invasion in E. coli when compared to wild-type Y. pestis Ail. Y. pseudotuberculosis YPIII Ail also had decreased binding to the Ail substrate fibronectin, relative to Y. pestis Ail in E. coli. When expressed in Y. pestis, there was a 30–50% decrease in adhesion and invasion depending on the substitution. Ail-mediated Yop delivery by both Y. pestis Ail and Y. pseudotuberculosis Ail were similar when expressed in Y. pestis, with only Ail-F126V giving a statistically significant reduction in Yop delivery of 25%. In contrast to results in E. coli and Y. pestis, expression of Ail in Y. pseudotuberculosis led to no measurable adhesion or invasion, suggesting the longer LPS of Y. pseudotuberculosis interferes with Ail cell-binding activity. Thus, host context affects the binding activities of Ail and both Y. pestis and Y. pseudotuberculosis Ail can mediate cell binding, cell invasion and facilitate Yop delivery.     

Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein

To relate the roles of Escherichia coli SSB in recombination in vivo and in vitro, we have studied the mutant proteins SSB-1 and SSB-113, the variant SSBc produced by chymotryptic cleavage, the partially homologous variant F SSB (encoded by the E. coli sex factor), and the protein encoded by gene 32 of bacteriophage T4. All of these, with the exception of SSB-1, augmented both the initial rate of homologous pairing and strand exchange promoted by RecA protein. From these and related observations, we conclude that SSB stimulates the initial formation of joint molecules by nonspecifically promoting the binding of RecA protein to single-stranded DNA; that SSB plays no role in synapsis of the RecA nucleoprotein filament with duplex DNA; that stimulation of strand exchange by SSB is similarly nonspecific; and that all members of the class of proteins represented by SSB, F SSB, and gene 32 protein may play equivalent roles in making single-stranded DNA more accessible to RecA protein.     

Localization of relaxin in the pig follicle during preovulatory development

The avidin-biotin immunoperoxidase method and antisera to purified porcine relaxin were used to localize relaxin in sections of follicles from pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG)-primed pigs during preovulatory development. Prepubertal pigs were treated i.m. with PMSG (750 IU) and 72 h later with hCG (500 IU) to induce follicular development and ovulation. Follicles were collected from untreated gilts or from gilts 24, 48, 60, 72, 84, 96, or 108 h after PMSG treatment. Light immunostaining in the theca interna was observed early in follicular development, at 48 and 60 h post-PMSG. At 72 h post-PMSG, relaxin immunostaining in the theca interna of the preovulatory follicle was more intense. After hCG treatment, the intense thecal immunostaining persisted and was apparent 84 and 96 h after PMSG. At about 6 h prior to expected ovulation (108 h post-PMSG), there was thinning of the follicle wall and a reduction in relaxin immunostaining in the theca interna. Immunoactive relaxin was not detected in follicles from untreated gilts, follicles 24 h post-PMSG, small healthy or atretic follicles, or in granulosa cells, theca externa or ovarian stroma, at any of the time points studied. These studies support the hypothesis that the theca interna is the primary source of follicular relaxin and provide further evidence for a paracrine role for relaxin in the ovulatory process.     

Cholesterol alterations in young dystrophic mice

Cholesterol and cholesteryl ester concentrations and cholesteryl ester fatty acid substituents have been measured during the first 10 weeks of life in tissues of normal and dystrophic mice. In normal Swiss and 129ReJ(+/?) mice the concentrations of both cholesterol and cholesteryl esters remain essentially constant in liver, increase in brain and fall sharply in both thigh (mixed fiber type muscles) and chest muscles (predominantly slow oxidative muscles) over this period. In all cases the concentration of free cholesterol exceeds that of esterified cholesterol. In dystrophic mice, similar patterns are found in brain and liver. In both thigh and chest muscles, however, the developmental pattern is significantly different. After an initial decrease the concentrations of cholesterol and cholesteryl esters increase rapidly with the largest increase occurring in the concentration of cholesteryl esters which by 10 weeks of age exceeds the concentration of cholesterol in chest muscle. During the same period the pattern of esterified fatty acids changes gradually in dystrophic tissues towards an increasing ratio of unsaturated/saturated fatty acids. By 10 weeks of age this ratio is significantly higher in dystrophic tissues than normal in all tissues tested.     

Differential production of steroids by dispersed granulosa and theca interna cells from developing preovulatory follicles of pigs

Dispersed granulosa and theca interna cells were recovered from follicles of prepubertal gilts at 36, 72 and 108 h after treatment with 750 i.u. PMSG, followed 72 h later with 500 i.u. hCG to stimulate follicular growth and ovulation. In the absence of aromatizable substrate, theca interna cells produced substantially more oestrogen than did granulosa cells. Oestrogen production was increased markedly in the presence of androstenedione and testosterone in granulosa cells but only to a limited extent in theca interna cells. The ability of both cellular compartments to produce oestrogen increased up to 72 h with androstenedione being the preferred substrate. Oestrogen production by the two cell types incubated together was greater than the sum produced when incubated alone. Theca interna cells were the principal source of androgen, predominantly androstenedione. Thecal androgen production increased with follicular development and was enhanced by addition of pregnenolone or by LH 36 and 72 h after PMSG treatment. The ability of granulosa and thecal cells to produce progesterone increased with follicular development and addition of pregnenolone. After exposure of developing follicles to hCG in vivo, both cell types lost their ability to produce oestrogen. Thecal cells continued to produce androgen and progesterone but no longer responded to LH in vitro. These studies indicate that several functional changes in the steroidogenic abilities of the granulosa and theca interna compartments occur during follicular maturation.     

Techniques for hand-rearing tree-shrews (Tupaia belangeri) from birth

In captivity, Tupaia belangeri (Thailand tree-shrew) frequently show aberrant patterns of maternal care which result in the death of offspring. In order to maximize the potential of the tree-shrew as an animal resource for experimental studies we have developed a program for hand-rearing tupaiidae from birth. Newborn tree-shrews were removed from mothers with a history of poor parental care to a nursery maintained under conditions of controlled relative humidity (70 ± 10%) and temperature (25 ± 1°C) on a 12 h dark: 12 h light cycle. The young tree-shrews were fed on a liquid formula until the eyes opened (Day 18–23) and for the subsequent 10 days on a transitional diet until they could feed themselves on solid food. Our hand-rearing protocol appears to conform to the natural weaning pattern of tupaiids. Food consumption during the liquid diet phase was linearly related to age and the increase in body weight in both sexes. The initial growth rate of male and female hand-reared tree-shrews was slower than that of maternally reared animals during the liquid diet phase. The growth rate accelerated subsequently and the body weight of hand-reared tree-shrews eventually reached that of maternally reared animals of the same sex. Various developmental changes occurred during the same period in artificially and naturally reared animals. In both hand-reared and maternally reared groups, the growth rate of male tree-shrews exceeded that of females from about Day 40 onward. The accelerated growth rate of male tree-shrews was in apparent association with an increase in androgen secretion at the onset of puberty. The high fecundity of tree-shrews in captivity reinforced with a program for hand-rearing the young make T belangeri a potential alternative to more conventional laboratory species in specific areas of biomedical research.     

Schistosoma mansoni adult microsomal antigens, a serologic reagent. I. Systematic fractionation, quantitation, and characterization of antigenic components

A systematic and quantitative search was conducted to identify and isolate a serologically pertinent antigen with high specific activity and low cross-reactivity from adult worms of Schistosoma mansoni. Adult worms of S. mansoni quickly thawed from liquid N2 temperatures were disrupted by controlled homogenization in isotonic buffered sucrose. Differential centrifugation of the homogenate yielded three particulate and one soluble fractions: the 480 x G pellet (nuclear), the 7650 x G pellet (mitochondrial), the 360,000 x G pellet (microsomal), and the 360,000 x G supernatant (cytosol). Quantitative analysis indicated a major concentration of specific antigenic activities in the microsomal fraction. Further purifications of the urea-solubilized, n-butanol-treated microsomal particles by gel filtration and ionic-exchange chromatography resulted in a microsomal antigen (MAMA) possessing high specific activity and low cross-reactivity. The final purification post-ionic exchange chromatograph showed a 30-fold increase of specific antigen activity over that of the cytosol fraction. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunotransfer blot by the "Western blot" technique (EITB) indicated specific antigenic activities in association with several different m.w. bands (heterogeneous m.w. by extrapolation = 4.3 to 11.9 x 10(6), 2.0 x 10(5), and 2.0 x 10(4) daltons) of the MAMA fraction. When compared with other reported serologic antigens, MAMA showed substantially higher specific activity and lower cross-reactivity.     

WNT-mediated relocalization of dishevelled proteins

Summary TheWnt family of proto-oncogenes encodes secreted signaling proteins that are required for mouse development. TheDrosophila Wnt homolog, thewingless (Wg) segment polarity gene, mediates a signal transduction pathway in which the downstream elements appear to be conserved through evolution. One such element, thedishevelled gene product, becomes hyperphosphorylated and translocates to the plasma membrane in response to Wg (Yanagawa et al., 1995). We report here that the mouseDishevelle-1 (Dvl-1) andDishevelled-2 genes encode proteins that are differentially localized inWnt-overexpressing PC12 cell lines (PC12/Wnt). WhereasDvl-1 andDvl-2 proteins are limited to the soluble fraction of parental PC12 cells, PC12/Wnt cells display a subset ofDvl-1 protein associated with the membrane andDvl-2 protein with the cytoskeletal fraction. These results suggest a conserved role forDvl inWnt/wg signal transduction.